Fig. 6

HA titer, median tissue culture infectious dose (TCID₅₀) and growth curve of recombinant viruses. Viruses were inoculated into chicken embryo allantoic cavity (9-day-old specific pathogen free chicken embryo, 0.25 μL each) and its Hemagglutination (HA) titer was determined. TCID₅₀ was determined through chicken embryo fibroblasts (CEF) and calculated by the Reed–Muench method. To determine growth curve, CEF were infected with each virus at MOI of 0.01 in M199 for 1 h. The infected cells were washed with PBS and then serum-free M199 was added. Cells were incubated at 37 °C under 5% CO2. The virus titers in the supernatant were monitored periodically by determination of TCID₅₀ in CEF. Each reaction was repeated for 3 times, and the titers were expressed as means ± standard deviation